Retinal ganglion cell (RGC) axons from each eye grow towards the brain and meet at the midline of the ventral diencephalon, establishing an X-shaped decussation, the optic chiasm. The navigation of retinal axons as they diverge to ipsi- and contralateral targets at the optic chiasm is a model for axon guidance at the CNS midline. The optic chiasm also patterns binocular vision, and if RGC divergence is mis-apportioned in inherited defects such as albinism, reduced visual acuity and strabismus ensue. The proposed analyses build on work in the last fifteen years on the identification of RGC behavior and the cellular/molecular cues during chiasm formation, recently highlighting how the uncrossed projection is established. Our new work shows that guidance factor ephrin-B2 is expressed in chiasm midline radial gila, and is selectively inhibitory to ventrotemporal (VT) RGCs, source of the uncrossed projection in mouse. EphB1, a receptor for ephrin-B2, is uniquely expressed in VT RGCs during the formation of the uncrossed projection. In the absence of EphB1, the uncrossed RGC projection fails to develop. Two sets of analyses will investigate this guidance system during RGC divergence. In Aim 1, studies will determine whether EphB1 expression is conserved in other vertebrates and primates, and whether EphB1 directs the projection of all uncrossed axons in the mouse, especially the early uncrossed RGCs arising from dorsocentral retina. Misexpression of EphB1 will test whether it is sufficient to confer sensitivity to ephrin-B2 and to convert contralateral to ipsilateral projections. In Aim 2, EphB1 protein will be localized on retinal axons, the site of EphB1 synthesis established (soma vs. growth cone), and the fate of receptor and ligand tracked after growth cone interactions with midline gila. Other experiments will characterize intracellular events such as calcium dynamics as RGC growth cones respond to ephrin midline cues. Such studies are important for understanding how visual pathways form, key for aiding RGC regeneration after perturbation to the visual system.